The ability of astrocytes to regulate the extracellular concentrations of neuroactive substances such as K+
and glutamate depends upon the presence of K+
channels in their membranes and the hyperpolarized membrane potential of these cells [4
]. We and others have previously shown that the highly hyperpolarized membrane potential of glial cells is largely due to the presence of Kir4.1 inward rectifying K+ channels [4
], although Kir4.1 channels are not the only channels contributing to the overall negative membrane potential of glia [4
]. Particularly in astrocytes, after knock-down [4
] or pharmacological blockade () of Kir4.1-containing channels, inward currents are dramatically reduced whereas robust outward currents remain under physiological K+
conditions. This current profile is consistent with the presence of tandem-pore domain K+
] in astrocytes.
In the present study, we used Western blot, electrophysiology, pharmacology and a glutamate clearance assay to determine the properties and the putative functional role of TREK-2 channels in astrocytes during experimental ischemia. We confirm that TREK-2 channels are present in cultured astrocytes under control conditions (as determined by I/V curve and pharmacology and temperature-sensitivity) and that these channels are functionally up-regulated during ischemic conditions. The ability of ischemia to increase TREK-2 levels is relatively selective as expression of several other potassium channels in astrocytes (TASK-1, TASK-3 and Kir4.1) remains unchanged.
After 2 hours of experimental ischemia, expression of TREK-2 protein was increased to 175% of control, suggesting that regulation of TREK-2 protein changes may occur at the translational and not transcriptonal level. There are a number of post-transcriptional mechanisms that could be involved: 1) regulation of translation by factors binding to the UTR of mRNA [34
], 2) liberation of TREK-2 mRNA from production bodies or p-bodies [35
] and/or 3) decreased degradation of protein [36
]. All of these processes would result in increased TREK-2 protein within the astrocyte. Alternatively, rapid changes in mRNA expression could account for these differences, although Xu et al.
] reported no changes in TREK-2 mRNA levels in rat hippocampus and cortex after 30 days of permanent bilateral carotid artery ligation.
We determined if this increased TREK-2 protein corresponded with an increase in functional TREK-2 channels in the astrocytic membrane. Using whole-cell voltage-clamp recording and increases in temperature to activate TREK-2 channels [28
], we found much larger outward currents in astrocytes exposed to ischemic conditions than in control astrocytes (). Furthermore, there was a much greater activation of outward currents recorded from “ischemic” astrocytes in response to increased temperature known to increase TREK-2 channel activity [28
]. This suggests that not only are TREK-2 protein levels increased in these astrocytes, but also that they are forming functional channels in the astrocytic membrane.
To determine if functional up-regulation of TREK-2 channels could help to protect neurons during ischemic events, we evaluated the ability of astrocytes to clear glutamate. Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system [37
]. Rapid removal of glutamate from the extracellular space is required for the survival and normal function of neurons. High-affinity Na+-dependent electrogenic transporters maintain low extracellular glutamate concentration. Although the glutamate transporters are expressed in both astrocytes and neurons, astrocytes are the cell type primarily responsible for glutamate uptake [39
]. Extracellular glutamate levels are increased following ischemia, hypoglycemia and trauma [41
] and if in vitro
extracellular glutamate levels increase >100 μM for longer than 5 min, neuronal death can occur [42
]. It has recently been shown that reactive astrocytes are neuroprotective during brain ischemia in vivo
and this neuroprotection is mediated, in part, by glutamate transport [43
Glutamate clearance depends upon glial cells having a hyperpolarized membrane potential [4
]. During ischemia when ATP is reduced, Kir4.1 channels should not be functioning optimally and unless compensatory changes in other K+ channels occur, the membrane potential of astrocytes will be depolarized resulting in decreased glutamate clearance.
Using a colorimetric assay to assess glutamate clearance, we found no significant difference in the ability of control astrocytes and astrocytes subjected to ischemic conditions to clear glutamate. The finding that glutamate clearance is comparable in both normal and ischemic astrocytes suggests that TREK-2 channel up-regulation may compensate during ischemic conditions. To test this, we determined the contribution of glutamate clearance due to TREK-2 channels in both the control and ischemic conditions. The most notable difference observed was the amount of clearance inhibited by 100 μM quinine; a concentration of quinine that blocks arachidonic acid sensitive K+
channels (some of 2P-domain channels including TREK-2) in astrocytes [11
], but not Kir4.1 channels (see results). This suggests a much greater contribution of TREK-2 channels to glutamate clearance by astrocytes exposed to ischemic conditions. Moreover, after blockade of TREK-2 channels in astrocytes subjected to ischemia, there is an apparent release of glutamate from the cells. Although the mechanism of this glutamate release is not known, it could perhaps be due to reverse transport by the glutamate transporter [45
] or release from hemichannels [46
] that are known to be opened during metabolic inhibition [47
]. Disregarding the mechanism, these data do suggest that TREK-2 channels help to rescue astrocytic buffering of glutamate.
In summary, TREK-2 channels are functionally up-regulated in astrocytes after ischemia. Taken together, these data suggest that up-regulation of TREK-2 channels may help maintain the membrane potential of astrocytes and lower extracellular glutamate and K+ concentrations during ischemia.